Focus on Salmonella

Salmonella Gram Stain

    The family Enterobacteriaceae contains many of the most familiar bacterial genera. Some, such as Salmonella, are of special interest to those involved in food production. These gram negative, facultatively anaerobic rod-shaped bacteria are pathogenic, causing thousands of cases of salmonellosis in the United States each year.  During 1996 and 1997, FoodNet found that Salmonella was the second most commonly detected pathogen in reported cases of foodborne illness. However, the large number of unreported cases make it difficult to determine how often salmonellosis really occurs.   Raw meats, poultry, eggs, milk and dairy products, and fish are among the most common sources of food-borne salmonellosis. Fruits, such as cantaloupe, are proving to be a common source as well.  While all species and strains of Salmonella are pathogenic, they are rarely lethal. Although, those with compromised immune systems and the elderly are at special risk. Severe forms of Salmonella infection exist, such as typhoid fever, which can be life threatening. However, the most common form of infection is the self-limiting Salmonella gastroenteritis. It is caused by ingestion of food or water containing sufficient numbers of the bacteria. In as few as six hours, nausea, vomiting, diarrhea, fever, and headache can occur. These symptoms often last one to two days.

     Pasteurization will destroy Salmonella, however its presence in products made with pasteurized milk (or other ingredients) demonstrates that improper processing or contamination after pasteurization can occur. These bacteria are commonly found in raw milk and therefore gain entrance into the dairy processing environment. Controlling environmental contamination and monitoring the presence of Salmonella is essential to prevent possible contamination of the product.  This is where the importance of sanitation comes in.

Detection of Salmonella

Salmonella on XLD agar

Salmonella on XLD agar

Salmonella VIA
     A negative or presumptive positive can be obtained in forty-eight hours using an ELISA (enzyme linked immunosorbent assay).   The day we receive the samples, they are pre-enriched in lactose/brilliant green broth for 18 to 22 hours at 35°C at a 1 to 10 dilution ( for example 400g/3600ml media). A series of transfers allows further enrichment. After the initial 18 to 22 hours pass, 0.1 ml is transferred for a 6-8 hour enrichment in Rappaport-Vassiliadis (RV) at 42°C. A second and final enrichment of 16 to 20 hours occurs in M broth. The Tecra ELISA can then be run. The Salmonella VIA uses specific monoclonal antibodies, which are adsorbed onto the microwells into which the samples are added. If Salmonella antigens are present in the sample, a complex is formed between the antibody and antigen. An enzyme labeled antibody (the conjugate) is added and will "sandwich" the antigen if present. The bound conjugate will cause a green color once the substrate is added. Therefore, if there is no Salmonella, there will be no green color.   If green color does appear, the absorbance must reach a minimum of 0.300 for the sample to be presumptive positive.   When we have a presumptive positive, it is confirmed by streaking from the retained M Broth and RV onto Hektoen enteric (HE) agar, Xylose lysine desoxycholate (XLD) agar, and Bismuth sulfite (BS) agar. Typical or suspect colonies of Salmonella are then streaked onto triple sugar iron agar (TSI) and lysine iron agar (LIA). The sample is cleared or confirmed based on the results on these two agar slants and if confirmed, serotyping begins.  However, on occasion we will have a sample reach 0.300 or slightly higher and fail to confirm.  With such a low absorbance, it is possible that a close relative of Salmonella is present, such as Citrobacter.  Or that dead Salmonella cells are present in the sample and therefore will not confirm but will trigger the reaction in the ELISA. 

How Salmonella is detected using the ELISA

  • Antibodies specific for Salmonella already in wells
  • If Salmonella antigens are present in the sample, they attach to the antibodies
  • Enzyme-labeled antibodies (conjugate) are added and complex with antigen if present
  • Substrate converted to a green color if conjugate is present    If absorbance is above 0.3,  sample is presumptive positive and undergoes confirmation    

References

AOAC International. 1995. Salmonella.  In FDA Bacteriological Manual.  8th Edition. AOAC International, Arlington, VA.

Frenzen P, Riggs T, Buzby J, Breuer T, Roberts T, Voetsch D, Reddy S, and the FoodNet Working Group.  "Salmonella Cost Estimate Update Using FoodNet Data."  Food Review 1999; 22 (2): 10-15.

Marshall, RT.  1992.  Standard Methods for the Examination of Dairy Products.  16th Ed.  American Public Health Association.  Washington DC.

Methods Manual.  Tecra Salmonella Visual Immunoassay for the Detection of Salmonella in Food and Food-related Samples.

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